肝细胞癌转移相关分泌蛋白的发现和初步验证

发布时间：2018-06-04 09:40

本文选题：肝癌 + 转移　；参考：《安徽医科大学》2015年硕士论文

【摘要】：肝细胞癌(hepatocellular carcinoma, HCC)是世界上发病率排名第七,死亡率排名第三的恶性肿瘤。在我国每年约有383,000人死于肝癌,大约占全世界肝癌死亡人数的50%,且逐年增加。肝癌的转移是临床治疗失败、致死率居高不下的主要因素。目前肝癌转移的分子机制尚不完全明确,仍没有可用于肝癌转移复发临床检测的生物标志物和有效的治疗靶标,发掘肝癌转移相关的重要蛋白具有重要的意义。肿瘤的转移涉及肿瘤细胞之间、肿瘤细胞和胞外基质环境之间的相互作用,包括胞外基质(extracellular matrix, ECM)重塑、免疫抑制、靶器官组织的损害和血管生成等过程。肿瘤分泌蛋白既可以自分泌、旁分泌的形式调控肿瘤发生发展的过程,也可直接进入血液、尿液、组织间隙液等体液中作为肿瘤转移标志物。蛋白质组学作为一种全局性、高通量的蛋白质分析策略,被广泛应用于以肿瘤为代表的多基因、多因素复杂疾病的发病机制研究。本课题将细胞培养条件下稳定同位素标记(stable isotope labeling with amino acids in cell culture,SILAC)定量蛋白质组学技术应用到肝癌分泌蛋白的分析之中,以期发现肝癌转移相关蛋白。研究选择背景相同、转移能力逐渐增高的三株肝癌细胞MHCC97L、 MHCC97H和HCCLM6作为分析材料,利用SILAC技术对三株细胞无血清培养上清中提取的分泌蛋白进行分析,共鉴定到1287个蛋白,其中分泌蛋白、膜蛋白和细胞膜连接蛋白共占总鉴定蛋白的18%。MHCC97H/MHCC97L中共鉴定到890个蛋白,有174个是差异蛋白(ratio±1.5倍),其中40个蛋白在转移能力较高的MHCC97H中上调,134个蛋白下调；在HCCLM6/MHCC97L中共鉴定到983个蛋白,有182个差异蛋白(ratio≥±1.5倍),其中67个蛋白在转移能力较高的HCCLM6中上调,115个蛋白下调。对上调蛋白进行生物过程分析,发现显著富集的前十位生物过程为细胞黏附、基因调控,核酸代谢,蛋白代谢,糖代谢、细胞分化、DNA代谢、细胞识别、生物聚合物修饰和脂类代谢。对上调蛋白进行通路分析,发现上调蛋白显著富集到溶酶体、聚糖降解和ECM-受体相互作用等通路。差异蛋白中有14个蛋白质的表达量随肝癌细胞转移能力增强而逐步上调,包括6种已知与肿瘤转移正相关的蛋白质如基质金属蛋白酶-1 (Matrix metalloproteinase-1, MMP1)、膜联蛋白A1 (Annexin A1, ANXA1)和角蛋白18(Keratin, type I cytoskeletal 18, KRT18)等,这也反映了我们数据的可靠性。另外还有4种蛋白质胶原蛋白六α-1链(Collagen alpha-1 (VI) chain, COL6A1)、光蛋白聚糖(Lumican, LUM)、N-乙酰葡糖胺-6-硫酸酯酶(N-acetylglucosamine-6-sulfatase, GNS)和前蛋白转化酶枯草溶菌素9(Proprotein convertase subtilisin/kexin type 9, PCSK9)未曾有与肝癌转移相关的报道,可作为候选蛋白进一步关注。对上调倍数较大的分泌蛋白COL6A1 (MHCC97H/MHCC97L=2.77、 HCCLM6/MHCC97L=4.23)进行进一步验证。通过Western Blot的方法,在正常肝细胞LO2、无转移能力的两株肝癌细胞系SK-Hep1和HepG2、以及三株转移能力依次升高的肝癌细胞系MHCC97L、MHCC97H、HCCLM6的分泌蛋白中检测COL6A1的蛋白含量。结果表明,COL6A1在LO2、SK-Hep1和HepG2分泌蛋白中没有被检测到,而该蛋白在转移能力依次升高的细胞系MHCC97L、MHCC97H和HCCLM6分泌蛋白中表达量依次升高。同时,COL6A1在LO2、SK-Hep1和HepG2的全细胞蛋白中也没有被检测到,而在MHCC97L、MHCC97H和HCCLM6的全细胞蛋白中表达量依次降低。上述结果表明COL6A在分泌蛋白中的含量与肝癌细胞的转移能力正相关,同时提示转移能力强的肝癌细胞可能具有更强的分泌COL6A1的能力。COL6A1广泛存在于胞外基质,涉及纤连蛋白和细胞的连接过程,并可与其它胶原蛋白(如胶原蛋白4,胶原蛋白1)连接,介导微纤维网络的形成。它与转移的相关性可能与改变ECM特性、促进细胞与ECM黏附和支持细胞运动有关。本研究发现并初步验证了COL6A1是与肝癌转移正相关的分泌蛋白,但其具体机制尚待进一步研究。为了进一步验证上述体外实验的结果,我们对转移能力不同的肝癌病人血清进行了Western Blot检测。遗憾的是由于目标蛋白在血中的丰度太低,Western Blot未能检测出COL6A1.我们下一步将积极寻找配对抗体用于临床血清样本的ELISA检测。除此之外,我们还对不同转移能力的人肝癌细胞系(HCCLM6和MHCC97)全细胞蛋白进行了iTRAQ (sobaric tags for relative and absolute quantitation)差异蛋白质组分析。共鉴定4804种蛋白质,4790种蛋白质有定量信息,发现103种差异蛋白(ratio≥±1.5),其中46种蛋白在高转移细胞HCCLM6中上调,57种下调。差异蛋白的细胞定位主要为细胞质和细胞膜,GO分析显著富集到细胞黏附等生物过程和蛋白结合等生物功能。发现了4种新的与肝癌转移相关的候选蛋白层粘连蛋白亚基a 5 (Laminin subunit alpha-5, LAMA5)、微管蛋白链β-2B(Tubulin beta-2B chain, TUBB2B)、着丝粒蛋白F (Centromere protein F, CENPF)和囊泡相关膜蛋白5 (Vesicle-associated membrane protein 5, VAMP5),为进一步肝癌转移机制研究提供了重要的数据参考。
[Abstract]:Hepatocellular carcinoma (HCC) is the seventh most malignant tumor in the world, and the mortality rate is third. In China, about 383000 people die of liver cancer every year, accounting for 50% of the death toll in the world, and the metastasis of liver cancer is the main factor of clinical treatment failure and high mortality. The molecular mechanism of the metastasis of liver cancer is still not completely clear. There is still no biomarker and effective target for the clinical detection of the metastasis of liver cancer. It is of great significance to explore the important proteins related to the metastasis of liver cancer. The metastasis of tumor involves the interaction between tumor cells, the tumor cell and the extracellular matrix environment, including the interaction between tumor cells and the extracellular matrix environment. Extracellular matrix (extracellular matrix, ECM) remodeling, immunosuppression, damage to target organ tissue and angiogenesis. Tumor secreting proteins can be autocrine, paracrine in the form of regulation of tumor development, and can also directly enter the blood, urine, interstitial fluid and other body fluids as tumor metastasis markers. As a global, high throughput protein analysis strategy, it is widely used in the study of the pathogenesis of multifactorial complex diseases represented by tumors. This topic uses stable isotope labeling (stable isotope labeling with amino acids in cell culture, SILAC) quantitative proteomics technology under cell culture. In the analysis of the secretory protein of liver cancer, the metastasis related protein of liver cancer was found. Three hepatoma cells, MHCC97L, MHCC97H and HCCLM6, which had the same background and increasing metastasis ability, were selected as the analytical materials. The secretory proteins extracted from the serum free culture supernatant of three cells were analyzed by SILAC technology, and 1287 of them were identified. Protein, in which the secretory protein, membrane protein and membrane connexin accounted for 890 proteins, 174 were differential proteins (ratio 1.5 times), of which 40 proteins were up regulated in MHCC97H with higher transfer ability and 134 proteins down; 983 proteins were identified in HCCLM6/MHCC97L and 18 in HCCLM6/MHCC97L. 2 differential proteins (ratio > 1.5 times), of which 67 proteins were up-regulated in HCCLM6 with higher transfer ability, and 115 proteins were downregulated. The biological process analysis of up regulated proteins showed that the first ten biological processes that were significantly enriched were cell adhesion, gene regulation, nucleic acid metabolism, egg white metabolism, sugar metabolism, cell differentiation, DNA metabolism, cell recognition and birth. The pathway analysis of up-regulated proteins showed that the up-regulated proteins were significantly enriched in lysosomes, chitosan degradation and ECM- receptor interaction. The expression of 14 proteins in the differential proteins increased gradually with the enhancement of the metastasis ability of hepatoma cells, including 6 eggs known to be positively related to tumor metastasis. White matter such as matrix metalloproteinase -1 (Matrix metalloproteinase-1, MMP1), membrane protein A1 (Annexin A1, ANXA1) and keratin 18 (Keratin, type I cytoskeletal 18, etc.), which also reflect the reliability of our data. There are also 4 kinds of protein collagen protein six alpha chain. Lumican, LUM, N- acetyl glucosamine -6- sulfate (N-acetylglucosamine-6-sulfatase, GNS) and preprotein invertase, wit lysozyme 9 (Proprotein convertase subtilisin/kexin type 9, PCSK9) have not been reported to be associated with metastasis of liver cancer. It can be used as a candidate protein for further attention. (MHCC97H/MHCC97L=2.77, HCCLM6/MHCC97L=4.23) for further validation. Through Western Blot, the content of COL6A1 protein was detected in the normal hepatocyte LO2, two liver cancer cell lines without metastasis, SK-Hep1 and HepG2, and the secretory proteins of the liver cancer cell line, which had the three metastatic ability in turn, in the secretory proteins of MHCC97H and HCCLM6. COL6A1 was not detected in the secretory proteins of LO2, SK-Hep1 and HepG2, and the protein expression in the cell lines, MHCC97L, MHCC97H and HCCLM6 secreting protein, in turn, increased in turn, and COL6A1 was not detected in LO2, SK-Hep1 and HepG2 whole cell proteins. The above results indicate that the content of COL6A in the secretory protein is positively related to the metastasis ability of the liver cancer cells, and suggests that the hepatoma cells with strong metastatic ability may have a stronger ability to secrete COL6A1,.COL6A1 is widely existed in the extracellular matrix, involving the connection between fibronectin and cells, and can be related to it. Its collagen (such as collagen 4, collagen 1) is connected to mediate the formation of a microfiber network. Its correlation with metastasis may be related to changes in ECM characteristics, promoting cell adhesion to ECM and supporting cell movement. This study found and preliminarily verified that COL6A1 is a positive secretory protein associated with metastasis of liver cancer, but its specific mechanism remains to be advanced. Step study. In order to further verify the results of the above in vitro experiments, we detected Western Blot in the serum of patients with different metastatic liver cancer. Unfortunately, the abundance of the target protein in the blood was too low, and Western Blot failed to detect COL6A1. we would be actively looking for the paired antibody for the clinical serum samples of ELISA next. In addition, we also carried out iTRAQ (sobaric tags for relative and absolute quantitation) differential proteomic analysis of human hepatoma cell lines (HCCLM6 and MHCC97) of human hepatocellular carcinoma cell lines (sobaric tags for relative and). 4804 proteins were identified, 4790 proteins had quantitative information, and 103 different proteins (ratio > 1.5) were found. The 46 proteins were up-regulated and 57 down regulated in the high transfer cell HCCLM6. The cell location of the differential proteins was mainly cytoplasm and cell membrane, GO analysis significantly enriched the biological processes of cell adhesion and other biological processes, and found 4 new candidate egg white laminin a 5 (Laminin subunit ALP) associated with the metastasis of liver cancer. Ha-5, LAMA5), microtubule protein chain beta -2B (Tubulin beta-2B chain, TUBB2B), centromere protein F (Centromere protein F, CENPF) and vesicular related membrane protein 5, provide important data reference for further study of the mechanism of liver cancer metastasis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

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